aSyn expression
Escherichia coli strain BL21(DE3) was transformed with pT7-7 aSyn WT vector43 from Addgene, (plasmid 36046) and plated onto a Luria broth (LB) agar plate containing ampicillin (100 μg ml−1) and 1 g l−1 glucose. A preculture in 5 ml of LB medium was inoculated with one clone and incubated at 37 °C under 200 rpm shaking for 4 h. Cells from the LB preculture were recovered by centrifugation (1,000g, 10 min) and used for inoculating 200 ml of LB medium. Cells were grown overnight at 37 °C under 200 rpm shaking and then diluted in 2 l of culture. Protein expression was induced by adding 0.5 mM isopropyl-β-d-thiogalactopyranoside during the exponential phase, evaluated at an optical density at 600 nm of 0.6. Cells were collected after 5 h of culture at 30 °C by centrifugation at 7,000g and 4 °C for 20 min (JLA 8.1000, Beckman Coulter), and the pellet was kept at −20 °C until purification.
aSyn purification
The pellets were resuspended in lysis buffer (10 mM Tris and 1 mM EDTA (pH 7.2) with protease inhibitor tablets (cOmplete, EDTA-free protease inhibitor cocktail, Roche)) and sonicated at 50% maximum energy, 30 s on and 30 s off for three rounds with a probe sonicator (Q-Sonica). The sonicated pellets were centrifuged at 20,000g for 30 min, and the supernatant was saved. The pH of the supernatant was then reduced to pH 3.5 using HCl, and the mixture was stirred at room temperature for 20 min and then centrifuged at 60,000g for 30 min. The pellets were discarded. The pH of the supernatant was then increased to pH 7.4 with NaOH and then dialysed against 20 mM Tris-HCl (pH 7.4) and 100 mM NaCl buffer before loading onto a 75 pg HiLoad 26/600 Superdex column equilibrated with the same buffer on the ÄKTA pure system. Monomeric fractions were collected and concentrated if needed by using the Vivaspin 15R 2 kDa cutoff concentrator (Sartorius Stedim). Purification fractions were analysed by polyacrylamide gel electrophoresis (PAGE) Tris-tricine 13% dying with ProBlue Safe Stain. The protein concentration was evaluated spectrophotometrically on the basis of the absorbance at 280 nm and an extinction coefficient of 5,960 M−1 cm−1. Quantification of the preparations using the Pierce chromogenic LAL kit indicated a low endotoxin (lipopolysaccharide, LPS) residual value of 0.03–0.06 EU μg−1 of recombinant protein.
aSyn fibrillization
Solutions of monomeric aSyn at 4 to 5 mg ml−1 in saline (H 2 O, 100 mM NaCl and 20 mM Tris-HCl pH 7.40) were sterilized by filtration through 0.22-μm Millipore single-use filters and stored in sterile 15 ml conical Falcon tubes at 4 °C. Sterilized stock was then distributed into safe-lock Biopur individually sterile-packaged 1.5 ml Eppendorf tubes as 500 μl aliquots and were seeded with 1% of 1B strain fibrils13,14,15. The tubes were cap-locked and additionally sealed with parafilm. All of the previous steps were performed aseptically in a particle-free environment under a microbiological safety laminar flow hood. The samples were loaded in a ThermoMixer (Eppendorf) in a 24-position 1.5 ml Eppendorf tube holder equipped with a heating lid. The temperature was set to 37 °C with continuous shaking at 2,000 rpm for 4 days. 1B templating of the fibrillization products was quality-checked using the fibrilloscope13,14,15.
Sonication
Before intracerebral injections, 1B aSyn fibril stocks (4 mg ml−1) were distributed in cap-locked, sterile 0.5 ml PCR tubes (Thermo Fisher Scientific). Sonication was performed at 25 °C in the Bioruptor Plus water bath sonicator (Diagenode) equipped with thermostatic control and an automated tube carousel rotator. The sonication power was set to high, and 10 cycles of 30 s on followed by 10 s off were applied. In agreement with our previous quantifications14, over 80% of the fibrils were 50-nm long or less after application of this protocol (not shown).
In vivo aSyn pathology
Adult 129SV (intrastriatal injections), C57BL/6 (nigral injections) and transgenic M83 (hemizygous, intrastriatal injections) mice were housed in a temperature-controlled (22 °C) and light-controlled environment under a 12 h–12 h light–dark cycle with forced ventilation (humidity below 50%) and with access to food and water ad libitum. All of the experimental procedures were conducted in accordance with the European Communities Council Directive (2010/63/EU) for care of laboratory animals. The study design was approved by the ethics committees of the University of Bordeaux, of the ANSES/Ecole Nationale Vétérinaire d’Alfort/Université Paris-Est Créteil and of the University of Salento, and authorized by the French Ministry of Higher Education and Research and by the Italian Ministry of Health (APAFIS 33147-2021091711598830 v6, APAFIS 37712-2022061615206629 v8, 0013178-P-17/05/2019). The mice (aged 6–8 weeks) unilaterally received 2 μl of sonicated aSyn fibrils 1B (4 mg ml−1) by stereotactic delivery at a flow rate of 0.4 µl min−1, and the pipette was left in place for 5 min after injection to avoid leakage. Delivery was performed within the right striatum (coordinates from bregma: anteroposterior (AP), −0.1; lateral (L), +2.5; dorsoventral (DV), +3.8), or above the right SN (coordinates from bregma: AP, −2.9; L, −1.3; DV, −4.5). WT mice (n = 20) and transgenic M83 hemizygous mice (n = 28) injected with either PBS, 1B fibrils or brain homogenates were euthanized after 6 weeks, 17 months, 24 months or when reaching the humane end points defined as death regarding the transgenic M83 mice, and were transcardially perfused with either Tris-buffered saline (pH 7.4) followed by 4% paraformaldehyde in PBS pH 7.4 at 4 °C, or with cacodylate buffer 0.1 M with 1 mM CaCl 2 (pH 7.4) followed by 4% paraformaldehyde plus 0.1% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4). Brains were subsequently postfixed in the same fixative. For standard neuropathology, the brains were paraffin embedded, and 10-µm-thick sections were cut using a rotative microtome (Leica). The sections of interest were deparaffinized and processed for epitope retrieval: the slides were immersed in citrate buffer pH 6 (Dako Agilent Technologies) and placed into a pressure cooker (Bio SB) at 114–121 °C for 10 min. After a cooling period of 20 min, the slides were washed twice for 5 min in PBS at room temperature. The slides were then processed for simple or double immunofluorescence using the following primary antibodies diluted at 1:500, or their combinations: EP1536Y (Abcam) or pSyn#64 (Wako) for detecting pS129aSyn-positive inclusions; LB509 for detecting human aSyn (Abcam, mouse monoclonal); anti-SOX10 (Abcam, rabbit monoclonal) for detecting OL nuclei; EP1532Y and anti-TH raised in chicken (anti-tyrosine hydroxylase, Abcam) for detecting the nigrostriatal tract; and anti-IBA1 (Abcam) for detecting microglia. DRAQ7 or DAPI were used to image the nuclei. The AlexaFluor-coupled secondary antibodies were from Thermo Fisher Scientific (Alexa 488, 568 and 674). The sections were acquired using a Pannoramic slide scanner (3D HISTECH, MM France) in epifluorescence mode, and multichannel fluorescence optical sections of the samples were performed (thickness, <0.8 µm) using either a Zeiss CD7 platform of a Leica SP5 laser-scanning confocal microscope equipped with a spectral detector, 488, 561 and 633 nm laser lines, a motorized xy stage and a mixed stepping motor/piezo z controller. The objective was ×40, and the z step size was set to 0.5 µm to produce stacks of 15 to 20 z planes. The pinhole was set to 1 airy unit. For 3D reconstructions and volume rendering/animations (corresponding to 360° tilt series of composite maximum pixel projections images), raw three-channel z-stack images were processed offline using Icy44 (v.2.4.0.0).
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